One of the most exciting advances in biomedical research is the discovery of small RNA- mediated gene regulation mechanisms. Our research supported by this R01 since 2002 has contributed significantly to this advancement: In the previous funding period, we discovered and characterized the function of mammalian Piwi/Argonaute proteins that are central to small RNA mechanisms. In the beginning of the current funding period, we further discovered that Piwi proteins bind to a complex class of more than 60,000 small non-coding RNAs that we called PIWI-interacting RNAs (piRNAs). Most piRNAs are encoded by intergenic junk DNA that is mostly heterochromatin. The discovery of piRNAs reveals a new and surprisingly complex dimension of biology, and was voted by the Science magazine as one of the ten Breakthroughs of 2006. Remarkably, Piwi proteins and piRNAs are abundantly expressed only in spermatogenic cells. We and others have shown that Piwi proteins play key roles in spermatogenesis in Drosophila, C. elegans, zebrafish, and mice. We have also shown that the overexpression of the human piwi gene (hiwi) is highly correlated to seminomas. To explore the mechanisms underlying the spermatogenic functions of Piwi proteins, we have recently demonstrated in Drosophila that Piwi proteins regulate the epigenetic state and transposon activity and in mice that MILI also binds to chromatin. Despite this exciting progress, the function of piRNAs has not been directly demonstrated in any system. The goal of this proposal is to demonstrate the function of piRNAs during spermatogenesis. Our working hypothesis is that piRNAs guide nuclear PIWI proteins to specific genomic loci for epigenetic regulation, which, in the case of transposons, lead to their silencing. To test this hypothesis and to explore the function of piRNAs, we propose to: (1) Determine the biological function of piRNA clusters during spermatogenesis. (2) Investigate the epigenetic effect of specific piRNAs to the genome. (3) Determine whether piRNAs are required for PIWI-targeting to genomic sequences. (4) Determine whether piRNAs are required for suppressing transposition.